Seed-preferred promoters from end genes

ABSTRACT

The invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions are nucleotide sequences for seed-preferred promoters isolated from genes for end1 and end2. A method for expressing a heterologous nucleotide sequence in a plant using the disclosed promoter sequences is provided. The method comprises transforming a plant cell to comprise a heterologous nucleotide sequence operably linked to one of the promoters of the invention and regenerating a stably transformed plant from the transformed plant cell.

This application claims the benefit of U.S. Application No. 60/098,230filed Aug. 28, 1998, which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of an operably linked promoter that is functionalwithin the plant host. Choice of the promoter sequence will determinewhen and where within the organism the heterologous DNA sequence isexpressed. Where continuous expression is desired throughout the cellsof a plant, constitutive promoters are utilized. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory element of choice. Where expression in specific tissuesor organs are desired, tissue-specific promoters may be used. That is,they may drive expression in specific tissues or organs. Suchtissue-specific promoters may be constitutive or inducible. In eithercase, additional regulatory sequences upstream and/or downstream fromthe core promoter sequence may be included in expression constructs oftransformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

Frequently it is desirable to have constitutive or inducible expressionof a DNA sequence in particular tissues or organs of a plant. Forexample, increased nutritional value of a plant might be accomplished bygenetic manipulation of the plant's genome to comprise a seed-preferredpromoter operably linked to a heterologous gene such that proteins withenhanced amino acid content are produced in the seed of the plant.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a tissue-specific promoter operably linked to anantisense nucleotide sequence, such that constitutive expression of theantisense sequence produces an RNA transcript that interferes withtranslation of the mRNA of the native DNA sequence.

Seed development involves embryogenesis and maturation events as well asphysiological adaptation processes that occur within the seed to insureprogeny survival. Developing plant seeds accumulate and storecarbohydrate, lipid, and protein that are subsequently used duringgermination. Expression of storage protein genes in seeds occursprimarily in the embryonic axis and cotyledons and in the endosperm ofdeveloping seeds but rarely in mature vegetative tissues. Generally, theexpression patterns of seed proteins are highly regulated. Thisregulation includes spatial and temporal regulation during seeddevelopment. A variety of proteins accumulate and decay duringembryogenesis and seed development and provide an excellent system forinvestigating different aspects of gene regulation as well as forproviding regulatory sequences for use in genetic manipulation ofplants.

Thus, isolation and characterization of seed-preferred promoters thatcan serve as regulatory regions for expression of heterologousnucleotide sequences of interest in a seed-preferred manner are neededfor genetic manipulation of plants.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel nucleotidesequence for modulating gene expression in a plant.

It is a further object of the present invention to provide an isolatedpromoter capable of driving transcription in a seed-preferred manner.

It is a further object of the present invention to provide a method ofimproved control of an endogenous or exogenous product in the seed of atransformed plant.

It is a further object of the present invention to provide a method forproviding useful changes in the phenotype of a seed of a transformedplant.

It is a further object of the present invention to provide a method forproducing a novel product in the seed of a transformed plant.

It is a further object of the present invention to provide a method forproducing a novel function in the seed of a transformed plant.

Therefore, in one aspect, the present invention relates to an isolatednucleic acid comprising an isolated promoter that is capable of drivingtranscription in a seed-preferred manner, wherein said promotercomprises a nucleotide sequence selected from the group consisting of:

a) sequences capable of driving expression of coding regions selectedfrom the group consisting of coding regions for end1 or end2;

b) a sequence comprising at least 40 contiguous nucleotides of thesequence set forth in either of SEQ ID NOS:1 or 4;

c) a sequence comprising a variant or fragment of the nucleotidesequence set forth in either of SEQ ID NOS: 1 or 4;

d) the nucleotide sequences set forth in SEQ ID NOS: 1 or 4;

e) a sequence that hybridizes to any one of SEQ ID NOS: 1 or 4 under lowstringency conditions; and

f) a nucleotide sequence complementary to a nucleotide sequence of (a)through (e).

In other aspects, the present invention relates to expression cassettescomprising the promoter operably linked to a nucleotide sequence,vectors containing the expression cassette, and plants stablytransformed with at least one expression cassette.

In a further aspect, the present invention relates to a method formodulating expression in the seed of a stably transformed plantcomprising the steps of (a) stransforming a plant cell with anexpression cassette comprising the promoter of the present inventionoperably linked to at least one nucleotide sequence; (b) growing theplant cell under plant growing conditions and (c) regenerating a stablytransformed plant from the plant cell wherein expression of thenucleotide sequence alters the phenotype of the seed.

Compositions and methods for regulating expression of heterologousnucleotide sequences in a plant are provided. Compositions are novelnucleotide sequences for seed-preferred plant promoters, moreparticularly transcriptional initiation regions isolated from the plantgenes end1 and end2 . A method for expressing a heterologous nucleotidesequence in a plant using the transcriptional initiation sequencesdisclosed herein is provided. The method comprises transforming a plantcell with a transformation vector that comprises a heterologousnucleotide sequence operably linked to one of the plant promoters of thepresent invention and regenerating a stably transformed plant from thetransformed plant cell. In this manner, the promoter sequences areuseful for controlling the expression of endogenous as well as exogenousproducts in a seed-preferred manner.

Downstream from and under the transcriptional initiation regulation ofthe seed-specific region will be a sequence of interest which willprovide for modification of the phenotype of the seed. Such modificationincludes modulating the production of an endogenous product, as toamount, relative distribution, or the like, or production of anexogenous expression product to provide for a novel function or productin the seed.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention nucleotide constructs are provided thatallow initiation of transcription in seed. Constructs of the inventioncomprise regulated transcriptional initiation regions associated withseed formation and seed tissues. Thus, the compositions of the presentinvention comprise novel nucleotide sequences for plant promoters,particularly seed-preferred promoters, more particularly endospermspecific promoters, for the genes end1 and end2. The end1 promoterdrives expression in transfer cells at an early stage in precursor cellsand continues expression into mature cells. The end2 promoter drivesexpression in aleurone cells.

The promoters for these genes may be isolated from the 5′ untranslatedregion flanking their respective transcription initiation sites. Methodsfor isolation of promoter regions are well known in the art.

The term “isolated” refers to material, such as a nucleic acid, whichis: (1) substantially or essentially free from components which normallyaccompany or interact with it as found in its naturally occurringenvironment; the isolated material optionally comprises material notfound with the material in its natural environment; or (2) if thematerial is in its natural environment, the material has beensynthetically (non-naturally) altered or produced by deliberate humanintervention to a composition and/or placed at a locus in the cell(e.g., genome or subcellular organelle) not native to a material foundin that environment. The alteration to yield the synthetic material canbe performed on the material within or removed from its natural state.

Methods are readily available in the art for the hybridization ofnucleic acid sequences. Promoter sequences from other plants may beisolated according to well-known techniques based on their sequencehomology to the promoter sequences set forth herein. In thesetechniques, all or part of the known promoter sequence is used as aprobe which selectively hybridizes to other sequences present in apopulation of cloned genomic DNA fragments (i.e. genomic libraries) froma chosen organism.

For example, the entire promoter sequence or portions thereof may beused as probes capable of specifically hybridizing to correspondingpromoter sequences. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such probes may be used toamplify corresponding promoter sequences from a chosen organism by thewell-known process of polymerase chain reaction (PCR). This techniquemay be used to isolate additional promoter sequences from a desiredorganism or as a diagnostic assay to determine the presence of thepromoter sequence in an organism.

Such techniques include hybridization screening of plated DNA libraries(either plaques or colonies; see e.g. Innis et al. (1990) PCR Protocols,A Guide to Methods and Applications, eds., Academic Press).

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

For purposes of defining the invention preferably low stringencyconditions are employed including hybridization with a buffer solutionof 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50 to 55° C. More preferably moderate stringency conditionsare employed including hybridization in 40 to 45% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Mostpreferably high stringency conditions are employed includinghybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a washin 0.1×SSC at 60 to 65° C. Hybridization times are not critical and canrange from about four hours to about sixteen hours.

An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) In general, sequences thatcorrespond to promoter sequences of the invention and hybridize to thepromoter sequence disclosed herein will be at least 50% homologous,preferably 60%, 70%, 80%, 85%, 90%, and even 95% homologous or more withthe disclosed sequences.

The promoter regions of the invention may be isolated from any plant,including, but not limited to corn (Zea mays), (Brassica napus, Brassicarapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), oats, barley,vegetables, ornamentals, and conifers. Preferably plants include corn,soybean, sunflower, safflower, oil seed Brassica, wheat, rice, barley,rye, alfalfa, and sorghum.

The coding sequence expressed by the promoters of the invention may beused for varying the phenotype of the seeds. Various changes inphenotype are of interest including modifying the fatty acid compositionin seeds, altering the starch or carbohydrate profile, altering theamino acid content of the seed, and the like. These results can beachieved by providing expression of heterologous or increased expressionof endogenous products in seeds. Alternatively, the results can beachieved by providing for a reduction of expression. of one or moreendogenous products, particularly enzymes or cofactors in the seed.These changes result in a change in phenotype of the transformed seed.

Genes of interest include, generally, those involved in oil, starch,protein, carbohydrate or nutrient metabolism as well as those affectingkernel size, sucrose loading, and the like. In particular, end1 may finduse in regulating the influx of nutrients. Both promoters are useful indisease resistance and in regulating expression of phytate genesparticularly to lower phytate levels in the seed.

General categories of genes of interest for the purpose of presentinvention include for example, those genes involved in information, suchas Zinc fingers, those involved in communication, such as kinases, andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes, for example, include genes encodingimportant traits for agronomics, insect resistance, disease resistance,herbicide resistance, and grain characteristics. It is recognized thatany gene of interest can be operably linked to the promoter of theinvention and expressed in the seed.

Important traits such as oil, starch and protein content can begenetically altered in addition to using traditional breeding methods.Modifications include altering the content of oleic acid, saturated andunsaturated oils, increasing levels of lysine and sulfur-containingamino acids and providing other essential amino acids, and alsomodification of starch. Hordothionin protein modifications are describedin WO94/16078; WO96/38562; WO96/08220; and U.S. Pat. No. 5,703,409issued Dec. 30, 1997, the disclosures of which are incorporated hereinin their entirety by reference. Another example is lysine and/or sulfurrich seed protein encoded by the soybean 2S albumin described inWO97/35023, and the chymotrypsin inhibitor from barley, Williamson etal. (1987) Eur. J. Biochem. 165:99-106, the disclosures of each areincorporated by reference. Derivatives of the following genes can bemade by site directed mutagenesis to increase the level of preselectedamino acids in the encoded polypeptide. For example, the gene encodingthe barley high lysine polypeptide (BHL), is derived from barleychymotrypsin inhibitor, WO98/20133, incorporated herein by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley et al. (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs;Applewhite, H. (ed.); American Oil Chemists Soc., Champaign,Ill.:497-502, incorporated herein in its entirety by reference), corn(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359, both incorporated herein in its entirety by reference) andrice (Musumura et al. (1989) Plant Mol. Biol. 12:123, incorporatedherein in its entirety by reference). Other agronomically importantgenes encode latex, Floury 2, growth factors, seed storage factors andtranscription factors.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteins,described in WO94/16078; WO96/38562; WO96/08220; and U.S. Pat. No.5,703,409; provide descriptions of modifications of proteins for desiredpurposes.

Commercial traits can also be encoded on a gene(s) which could alter orincrease for example, starch for the production of paper, textiles, andethanol, or provide expression of proteins with other commercial uses.Another important commercial use of transformed plants is the productionof polymers and bioplastics such as described in U.S. Pat. No. 5,602,321issued Feb. 11, 1997. Genes such as B-ketothiolase, PHBase(polyhydroxyburyrate synthase) and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol 170(12):5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof seed proteins, particularly modified seed proteins having improvedamino acid distribution to improve the nutrient value of the seed can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis endotoxin genes(U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881;Geiser etal. (1986) Gene 48:109); lectins (Van Damme et al. (1994) PlantMol. Biol. 24:825); and the like.

Genes encoding disease resistance traits may include detoxificationgenes, such as against fumonosin (U.S. patent application Ser. No.08/484,815 filed Jun. 7, 1995); avirulence (avr) and disease resistance(R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993)Science 262:1432; Mindrinos et al. (1994) Cell 78:1089; and the like.

Agronomic traits in seeds can be improved by altering expression ofgenes that affect the response of seed growth and development duringenvironmental stress, Cheikh-N et al (1994) Plant Physiol. 106(1):45-51)and genes controlling carbohydrate metabolism to reduce kernel abortionin maize, Zinselmeier et al. (1995) Plant Physiol. 107(2):385-391.

As noted, the heterologous nucleotide sequence operably linked to one ofthe promoters disclosed herein may be an antisense sequence for atargeted gene. By “antisense DNA nucleotide sequence” is intended asequence that is in inverse orientation to the 5′-to-3′ normalorientation of that nucleotide sequence. When delivered into a plantcell, expression of the antisense DNA sequence prevents normalexpression of the DNA nucleotide sequence for the targeted gene. Theantisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing to the endogenous messengerRNA (mRNA) produced by transcription of the DNA nucleotide sequence forthe targeted gene. In this case, production of the native proteinencoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Thus the promoter sequences disclosed herein may beoperably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant seed.

By “promoter” or “transcriptional initiation region” is intended aregulatory region of DNA usually comprising a TATA box capable ofdirecting RNA polymerase II to initiate RNA synthesis at the appropriatetranscription initiation site for a particular coding sequence. Apromoter may additionally comprise other recognition sequences generallypositioned upstream or 5′ to the TATA box, referred to as upstreampromoter elements, which influence the transcription initiation rate. Itis recognized that having identified the nucleotide sequences for thepromoter regions disclosed herein, it is within the state of the art toisolate and identify further regulatory elements in the 5′ untranslatedregion upstream from the particular promoter regions identified herein.Thus the promoter regions disclosed herein are generally further definedby comprising upstream regulatory elements such as those responsible fortissue and temporal expression of the coding sequence, enhancers and thelike. In the same manner, the promoter elements which enable expressionin the desired tissue such as the seed can be identified, isolated, andused with other core promoters to confirm seed-preferred expression.

The regulatory sequences of the present invention, when operably linkedto a heterologous nucleotide sequence of interest and inserted into atransformation vector, enable seed-preferred expression of theheterologous nucleotide sequence in the seeds of a plant stablytransformed with this vector.

By “seed-preferred” is intended expression in the seed, including atleast one of embryo, kernel, pericarp, endosperm, nucellus, aleurone,pedicel, and the like.

By “heterologous nucleotide sequence” is intended a sequence that is notnaturally occurring with the promoter sequence. While this nucleotidesequence is heterologous to the promoter sequence, it may be homologous,or native, or heterologous, or foreign, to the plant host.

It is recognized that the promoters may be used with their native codingsequences to increase or decrease expression resulting in a change inphenotype in the transformed seed.

The isolated promoter sequences of the present invention can be modifiedto provide for a range of expression levels of the heterologousnucleotide sequence. Thus, less than the entire promoter regions may beutilized and the ability to drive seed-preferred expression retained.However, it is recognized that expression levels of mRNA may bedecreased with deletions of portions of the promoter sequences.Generally, at least about 20 nucleotides of an isolated promotersequence will be used to drive expression of a nucleotide sequence.

It is recognized that to increase transcription levels enhancers may beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like.

Modifications of the isolated promoter sequences of the presentinvention can provide for a range of expression of the heterologousnucleotide sequence. Thus, they may be modified to be weak promoters orstrong promoters. Generally, by “weak promoter” is intended a promoterthat drives expression of a coding sequence at a low level. By “lowlevel” is intended at levels of about 1/10,000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts. conversely, astrong promoter drives expression of a coding sequence at a high. level,or at about 1/10 transcripts to about 1/00 transcripts to about 1/1,000transcripts.

The nucleotide sequences for the promoters of the present invention maybe the naturally occurring sequences or sequences having substantialhomology. By “substantial homology” is intended a sequence exhibitingsubstantial functional and structural equivalence with the naturallyoccurring sequence. Any structural differences between substantiallyhomologous sequences do not effect the ability of the sequence tofunction as a promoter as disclosed in the present invention. Thus,sequences having substantial sequence homology with the sequence of aparticular seed-preferred promoter of the present invention will directseed-preferred expression of an operably linked heterologous nucleotidesequence. Two promoter nucleotide sequences are considered substantiallyhomologous when they have at least about 70%, preferably at least about80%, more preferably at least about 90%, still more preferably at leastabout 95% sequence homology. Substantially homologous sequences of thepresent invention include variants of the disclosed sequences such asthose that result from site-directed mutagenesis, as well assynthetically derived sequences.

Substantially homologous sequences of the present invention also referto those fragments of a particular promoter nucleotide sequencesdisclosed herein that operate to promote the seed-preferred expressionof an operably linked heterologous nucleotide sequence. These fragmentswill comprise at least about 20 contiguous nucleotides, preferably atleast about 50 contiguous nucleotides, more preferably at least about 75contiguous nucleotides, even more preferably at least about 100contiguous nucleotides of the particular promoter nucleotide sequencedisclosed herein. The nucleotides of such fragments will usuallycomprise the TATA recognition sequence of the particular promotersequence. Such fragments may be obtained by use of restriction enzymesto cleave the naturally occurring promoter nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring promoter DNA sequence; or may be obtained throughthe use of PCR technology. See particularly, Mullis et al. (1987)Methods Enzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology(Stockton Press, New York). Again, variants of these promoter fragments,such as those resulting from site-directed mutagenesis, are encompassedby the compositions of the present invention.

Nucleotide sequences comprising at least about 40 contiguous sequencesof the sequences set forth in SEQ ID NOS: 1 and 4 are encompassed. Thesesequences may be isolated by hybridization, PCR, and the like. Suchsequences encompass fragments capable of driving seed-preferredexpression, fragments useful as probes to identify similar sequences, aswell as elements responsible for temporal or tissue specificity.Biologically active variants of the promoter sequences are alsoencompassed by the method of the present invention. Such variants shouldretain promoter activity, particularly the ability to drive expressionin seed or seed tissues. Biologically active variants include, forexample, the native promoter sequences of the invention having one ormore nucleotide substitutions, deletions or insertions. Promoteractivity may be measured by Northern blot analysis, reporter activitymeasurements when using transcriptional fusions, and the like. See, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.),herein incorporated by reference.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “percentage of sequenceidentity”, and (d) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length promoter sequence, or the complete promoter sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length and optionally can be30, 40, 50, 100, or more contiguous nucleotides in length. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443; by computerized implementations of these algorithms,including, but not limited to: GAP, BESTFIT, BLAST, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG)(575 Science Drive, Madison, Wis.). An example of the BLAST family ofprograms, which can be used to search database sequence similarity forthe purposes of this invention, includes BLASTN program for nucleotidequery sequences against nucleotide sequence dataset. See, Ausubel etal., eds. (1995) Current Protocols in Molecular Biology, Chapter 19(Greene Publishing and Wiley-Interscience, New York).

The BLAST homology alignment algorithm is useful for comparing fragmentsof the reference nucleotide or amino acid sequence to sequences frompublic databases. It is then necessary to apply a method of aligning thecomplete reference sequence against the complete public sequence toestablish a % identity (in the case of polynucleotides ) or % similarity(in the case of polypeptides). The GAP algorithm is such a method.

GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the. gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater. Unlessotherwise stated, for purposes of the invention, the preferred method ofdetermining percent sequence identity is by the GAP version 10 algorithmusing default parameters.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

(d) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters.

Another indication that nucleotide sequences are substantially identicalis if two nucleic acid molecules hybridize to each other under stringentconditions. Generally, stringent temperature conditions are selected tobe about 5° C. to about 2° C. lower than the melting point (T_(m)) forthe specific sequence at a defined ionic strength and pH. Thedenaturation or melting of DNA occurs over a narrow temperature rangeand represents the disruption of the double helix into its complementarysingle strands. The process usually is characterized by the temperatureof the midpoint of transition, T_(m), which is sometimes described asthe melting temperature. Formulas are available in the art for thedetermination of melting temperatures. Typically, stringent washconditions are those in which the salt concentration is about 0.02 molarat pH 7 and the temperature is at 50, 55, or 60° C.

The nucleotide sequences for the seed-preferred promoters disclosed inthe present invention, as well as variants and fragments thereof, areuseful in the genetic manipulation of any plant when operably linkedwith a heterologous nucleotide sequence whose expression is to becontrolled to achieve a desired phenotypic response. By “operablylinked” is intended the transcription or translation of the heterologousnucleotide sequence is under the influence of the promoter sequence. Inthis manner, the nucleotide sequences for the promoters of the inventionmay be provided in expression cassettes along with heterologousnucleotide sequences for expression in the plant of interest, moreparticularly in the seed of the plant.

Such expression cassettes will comprise a transcriptional initiationregion comprising one of the promoter nucleotide sequences of thepresent invention, or variants or fragments thereof, operably linked tothe heterologous nucleotide sequence whose expression is to becontrolled by the seed-preferred promoters disclosed herein. Such anexpression cassette is provided with a plurality of restriction sitesfor insertion of the nucleotide sequence to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

The expression cassette will include in the 5′-to-3′ direction oftranscription, a transcriptional and translational initiation region, aheterologous nucleotide sequence of interest, and a transcriptional andtranslational termination region functional in plants. The terminationregion may be native with the transcriptional initiation regioncomprising one of the promoter nucleotide sequences of the presentinvention, may be native with the DNA sequence of interest, or may bederived from another source. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also, Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

The expression cassette comprising the promoter sequence of the presentinvention operably linked to a heterologous nucleotide sequence may alsocontain at least one additional nucleotide sequence for a gene to becotransformed into the organism. Alternatively, the additionalsequence(s) can be provided on another expression cassette.

Where appropriate, the heterologous nucleotide sequence whose expressionis to be under the control of the promoter sequence of the presentinvention and any additional nucleotide sequence(s) may be optimized forincreased expression in the transformed plant. That is, these nucleotidesequences can be synthesized using plant preferred codons for improvedexpression. Methods are available in the art for synthesizingplant-preferred nucleotide sequences. See, for example, U.S. Pat. Nos.5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of theheterologous nucleotide sequence may be adjusted to levels average for agiven cellular host, as calculated by reference to known genes expressedin the host cell. When possible, the sequence is modified to avoidpredicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Nat. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example,TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader(Maize Dwarf Mosaic Virus) (Virology 154:9-20); human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991) Nature353:90-94); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);tobacco mosaic virus leader (TMV) (Gallie et al. (1989) MolecularBiology of RNA, pages 237-256); and maize chlorotic mottle virus leader(MCMV) (Lommel et al. (1991) Virology 81:382-385). See also Della-Cioppaet al. (1987) Plant Physiology 84:965-968. Other methods known toenhance translation and/or mRNA stability can also be utilized, forexample, introns, and the like.

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence directed to a particularorganelle, particularly the plastid, amyloplast or vacuole, or to theendoplasmic reticulum, or secreted at the cell's surface orextracellularly, the expression cassette may further comprise a codingsequence for a transit peptide. Such transit peptides are well known inthe art and include, but are not limited to, the transit peptide for theacyl carrier protein, the small subunit of RUBISCO, plant EPSP synthase,and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, for example,transitions and transversions, may be involved.

Reporter genes or selectable marker genes may be included in theexpression cassette. Examples of suitable reporter genes known in theart can be found in, for example, Jefferson et al. (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer AcademicPublishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737;Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques19:650-655; and Chiu et al. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate(Herrera Estrella et al. (1983) Nature 303:209-213; Meijer et al. (1991)Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al. (1985) PlantMol. Biol. 5:103-108; Zhijian et al. (1995) Plant Science 108:219-227);streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res.5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176);sulfonamide (Guerineau et al. 1990) Plant Mol. Biol. 15:127-136);bromoxynil (Stalker et al. (1988) Science 242:419-423); glyphosate (Shawet al. (1986) Science 233:478-481); phosphinothricin (DeBlock etal.(1987) EMBO J. 6:2513-2518).

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to, examples such as GUS (b-glucoronidase; Jefferson(1987) Plant Mol. Biol. Rep. 5:387), GFP (green florescence protein;Chalfie et al. (1994) Science 263:802), luciferase (Riggs et al. (1987)Nucleic Acids Res. 15(19):8115 and Luehrsen et al. (1992) MethodsEnzymol. 216:397-414) and the maize genes encoding for anthocyaninproduction (Ludwig et al. (1990) Science 247:449).

The expression cassette comprising the particular promoter sequence ofthe present invention operably linked to a heterologous nucleotidesequence of interest can be used to transform any plant. In this manner,genetically modified plants, plant cells, plant tissue, seed, and thelike can be obtained.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat No.5,563,055; Zhao et al. WO US98/01268), direct gene transfer (Paszkowskiet al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration(see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.(1995) “Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabeet al. (1988) Biotechnology 6:923-926). Also see Weissinger et al.(1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) ParticulateScience and Technology 5:27-37 (onion); Christou et al. (1988) PlantPhysiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet 96:319-324(soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein etal. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al.(1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855;Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al.(1995) “Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having seed-preferred expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that seed-preferred expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure seed-preferred expression of the desired phenotypiccharacteristic has been achieved.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Promoter regions for the maize genes end1 and end2 were isolated frommaize plants and cloned. These genes were selected as sources ofseed-preferred promoters based on the spatial expression of their geneproducts. The method for their isolation is described below.

EXAMPLE 1

Isolation of Promoter Sequences

The procedure for promoter isolation is described in the User Manual forthe Genome Walker kit sold by Clontech Laboratories, Inc., Palo Alto,Calif. Genomic DNA from maize line A63 was prepared by grinding10-day-old seedling leaves in liquid nitrogen, and the DNA prepared asdescribed by Chen and Dellaporta (1994) in The Maize Handbook, ed.Freeling and Walbot (Springer-Verlag, Berlin) with a few minormodifications. Precipitated DNA was recovered using an inoculation loopand transferred to a 1.5 ml eppendorf tube containing 500 μl of TE(10 mMTris pH 8.0, 1 mM EDTA). The DNA was allowed to dissolve at roomtemperature for 15 minutes, phenol extracted and 2-propanol precipitatedin 700 μl. The precipitate was recovered and washed with 70% ethanol.The DNA was then placed in a clean 1.5 ml eppendorf tube to air dry andresuspended in 200 l of TE. RNase A was added to 10 μg/ml and themixture was incubated at 37° C. for several hours. The DNA was thenextracted once with phenol-chloroform, then chloroform, then ethanolprecipitated and resuspended in TE. The DNA was then used exactly asdescribed in the Genome Walker User Manual (Clontech PT3042-1 versionPR68687). Briefly, the DNA was digested separately with restrictionenzymes DraI, EcoRV, PvuII, ScaI, and StuI, all blunt-end cutters. TheDNA was extracted with phenol, then chloroform, then ethanolprecipitated. The Genome Walker adapters were ligated onto the ends ofthe restricted DNA. The resulting DNA is referred to as DL1-DL5,respectively.

For isolation of specific promoter regions, two nonoverlappinggene-specific primers (27-30 bp in length) were designed from the 5′ endof the maize genes identified from sequence databases. The primers weredesigned to amplify the region upstream of the coding sequence, i.e. the5′ untranslated region and promoter of the chosen gene. The sequence ofthe primers are given below for each promoter described. The first roundof PCR was performed on each DNA sample (DL1-5) with Clontech primer AP1(SEQ ID NO:9) and the gene-specific primer (gsp)1 with the sequencesshown in SEQ ID NOS: 2 and 5.

PCR was performed in a model PTC-100 thermal cycler with HotBonnet fromMJ Research (Watertown, Mass.) using reagents supplied with the GenomeWalker kit. The following cycle parameters were used: 7 cycles of 94° C.for 2 seconds, then 72° C. for 3 minutes, followed by 32 cycles of 94°C. for 2 seconds and 67° C. for 3 minutes. Finally, the samples wereheld at 67° C. for 4 minutes and then at 4° C. until further analysis.

As described in the User Manual, the DNA from the first round of PCR wasthen diluted and used as a template in a second round of PCR using theClontech AP2 primer (SEQ ID NO:10) and gene-specific primer (gsp)2 withthe sequences shown in SEQ ID NOS: 3 and 6.

The cycle parameters for the second round were: 5 cycles of 94° C. for 2seconds, then 72° C. for 3 minutes. Finally, the samples were held at67° C. for 4 minutes and then held at 4° C. Approximately 10 μl of eachreaction were run on a 0.8% agarose gel, and bands (usually 500 bp orlarger) were excised, purified with the Sephaglas BandPrep kit(Pharmacia, Piscataway, N.J.) and cloned into the TA vector pCR2.1(Invitrogen, San Diego, Calif.). Clones were sequenced for verification.

EXAMPLE 2

Expression Data Using Promoter Sequences

Three promoter::GUS fusion constructs were prepared by the methodsdescribed below. All vectors were constructed using standard molecularbiology techniques (Sambrook et al., Supra). A reporter gene and aselectable marker gene for gene expression and selection was insertedbetween the multiple cloning sites of the pBluescript cloning vector(Stratagene Inc., 11011 N. Torrey Pines Rd., La Jolla, Calif.). Thereporter gene was the β-glucuronidase (GUS) gene (Jefferson, R. A. etal., 1986, Proc. Nat. Acad. Sci. (USA) 83:8447-8451) into whose codingregion was inserted the second intron from the potato ST-LS1 gene(Vancanneyt et al., Mol. Gen. Genet. 220:245-250, 1990), to produceintron-GUS, in order to prevent expression of the gene in Agrobacterium(see Ohta, S. et al., 1990, Plant Cell Physiol. 31(6):805-813). Therespective promoter regions were ligated in frame to sites 5′ to the GUSgene. A fragment containing bases 2 to 310 from the terminator of thepotato proteinase inhibitor (pinII) gene (An et al., Plant Cell1:115-122, 1989) was blunt-end ligated downstream of the GUS codingsequence, to create the GUS expression cassette. The 3′ end of theterminator carried a NotI restriction site.

The promoter fusion end1::GUS::pinII was constructed using theGUS::pinII plasmid digested with BamHI, filled in with Klenow, anddigested with NcoI. The promoter was isolated from the TOPOTA vector bydigestion with AvaI, filled in with Klenow, and then digestion withNcoI. The fragment was ligated into the digested expression cassette,and successful subcloning was confirmed by restriction digest andsequencing.

The Agrobacterium transformation plasmids were constructed by insertingthe GUS expression cassette as a HindIII/NotI fragment and the BARexpression cassette as a NotI/SacI fragment between the right and leftT-DNA borders in pSB11 at HindIII and SacI sites. The GUS cassette wasinserted proximal to the right T-DNA border. The plasmid pSB11 wasobtained from Japan Tobacco Inc. (Tokyo, Japan). The construction ofpSB11 from pSB21 and the construction of pSB21 from starting vectors isdescribed by Komari et al. (1996, Plant J. 10:165-174). The T-DNA of theplasmids were integrated into the superbinary plasmid pSB1 (Saito etal., EP 672 752 A1) by homologous recombination between the twoplasmids. The plasmid pSB1 was also obtained from Japan Tobacco Inc. E.coli strain HB101 containing the expression cassettes was mated withAgrobacterium strain LBA4404 harboring pSB1 to create the cointegrateplasmid in Agrobacterium using the method of Ditta et al., (Proc. Natl.Acad. Sci. USA 77:7347-7351, 1980). Successful recombination wasverified by a SaII restriction digest of the plasmid.

EXAMPLE 3

Transformation and Regeneration of Maize Callus via Agrobacterium

Preparation of Agrobacterium Suspension

Agrobacterium is streaked out from a −80° frozen aliquot onto a platecontaining PHI-L medium and cultured at 28° C. in the dark for 3 days.PHI-L media comprises 25 ml/l Stock Solution A, 25 ml/l Stock SolutionB, 450.9 ml/l Stock Solution C and spectinomycin (Sigma Chemicals) addedto a concentration of 50 mg/l in sterile ddH2O (stock solution A: K2HPO460.0 g/l, NaH2PO4 20.0 g/l, adjust pH to 7.0 w/KOH and autoclave; stocksolution B: NH4Cl 20.0 g/l, MgSO4.7H2O 6.0 g/l, KCl 3.0 g/l, CaCl2 0.20g/l, FeSO4.7H2O 50.0 mg/l, autoclave; stock solution C: glucose 5.56g/l, agar 16.67 g/l (#A-7049, Sigma Chemicals, St. Louis, Mo.) andautoclave).

The plate can be stored at 4° C. and used usually for about 1 month. Asingle colony is picked from the master plate and streaked onto a platecontaining PHI-M medium [yeast extract (Difco) 5.0 g/l; peptone(Difco)10.0 g/l; NaCl 5.0 g/l; agar (Difco) 15.0 g/l; pH 6.8, containing50 mg/L spectinomycin] and incubated at 28° C. in the dark for 2 days.

Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l,Eriksson's vitamin mix (1000X, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l (Sigma); 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma) 1.5 mg/l;L-proline (Sigma) 0.69 g/l; sucrose (Mallinckrodt) 68.5 g/l; glucose(Mallinckrodt) 36.0 g/l; pH 5.2] for the PHI basic medium system, orPHI-I [MS salts (GIBCO BRL) 4.3 g/l; nicotinic acid (Sigma) 0.5 mg/l;pyridoxine.HCl (Sigma) 0.5 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol(Sigma) 0.10 g/l; vitamin assay casamino acids (Difco Lab) 1.0 g/l; 2,4-D 1.5 mg/l; sucrose 68.50 g/l; glucose 36.0 g/l; adjust pH to 5.2w/KOH and filter-sterilize] for the PHI combined medium system and 5 mlof 100 mM (3′-5′-Dimethoxy4′-hydroxyacetophenone, Aldrich chemicals) areadded to a 14 ml Falcon tube in a hood. About 3 full loops (5 mm loopsize) Agrobacterium is collected from the plate and suspended in thetube, then the tube is vortexed to make an even suspension. One ml ofthe suspension is transferred to a spectrophotometer tube and the OD ofthe suspension adjusted to 0.72 at 550 nm by adding either moreAgrobacterium or more of the same suspension medium, for anAgrobacterium concentration of approximately 0.5×109 cfu/ml to 1×109cfu/ml. The final Agrobacterium suspension is aliquoted into 2 mlmicrocentrifuge tubes, each containing 1 ml of the suspension. Thesuspensions are then used as soon as possible.

Embryo Isolation, Infection and Co-cultivation

About 2 ml of the same medium (here PHI-A or PHI-I) used for theAgrobacterium suspension are added into a 2 ml microcentrifuge tube.Immature embryos are isolated from a sterilized ear with a sterilespatula (Baxter Scientific Products S1565) and dropped directly into themedium in the tube. A total of about 100 embryos are placed in the tube.The optimal size of the embryos is about 1.0-1.2 mm. The cap is thenclosed on the tube and the tube vortexed with a Vortex Mixer (BaxterScientific Products S8223-1) for 5 sec. at maximum speed. The medium isremoved and 2 ml of fresh medium are added and the vortexing repeated.All of the medium is drawn off and 1 ml of Agrobacterium suspension isadded to the embryos and the tube vortexed for 30 sec. The tube isallowed to stand for 5 min. in the hood. The suspension of Agrobacteriumand embryos was poured into a Petri plate containing either PHI-B medium[CHU(N6) basal salts (Sigma C-1416) 4.0 g/l; Eriksson's vitamin mix(1000X, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; silver nitrate 0.85 mg/l; gelrite (Sigma) 3.0 g/l;sucrose 30.0 g/l; acetosyringone 100 mM; pH 5.8], for the PHI basicmedium system, or PHI-J medium [MS Salts 4.3 g/l; nicotinic acid 0.50mg/l; pyridoxine HCl 0.50 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol100.0 mg/l; 2, 4-D 1.5 mg/l; sucrose 20.0 g/l; glucose 10.0 g/l;L-proline 0.70 g/l; MES (Sigma) 0.50 g/l; 8.0 g/l agar (Sigma A-7049,purified) and 100 mM acetosyringone with a final pH of 5.8 for the PHIcombined medium system. Any embryos left in the tube are transferred tothe plate using a sterile spatula. The Agrobacterium suspension is drawnoff and the embryos placed axis side down on the media. The plate issealed with Parafilm tape or Pylon Vegetative Combine Tape (productnamed “E.G.CUT” and is available in 18 mm×50 m sections; Kyowa Ltd.,Japan) and incubated in the dark at 23-25° C. for about 3 days ofco-cultivation.

Resting, Selection and Regeneration Steps

For the resting step, all of the embryos are transferred to a new platecontaining PHI-C medium [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l;Eriksson's vitamin mix (1000X Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer(Sigma) 0.5 g/l; agar (Sigma A-7049, purified) 8.0 g/l; silver nitrate0.85 mg/l; carbenicillin 100 mg/l; pH 5.8]. The plate is sealed withParafilm or Pylon tape and incubated in the dark at 28° C. for 3-5 days.

Longer co-cultivation periods may compensate for the absence of aresting step since the resting step, like the co-cultivation step,provides a period of time for the embryo to be cultured in the absenceof a selective agent. Those of ordinary skill in the art can readilytest combinations of co-cultivation and resting times to optimize orimprove the transformation frequency without undue experimentation.

For selection, all of the embryos are then transferred from the PHI-Cmedium to new plates containing PHI-D medium, as a selection medium,[CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l; Eriksson's vitamin mix(1000X, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer 0.5 g/l; agar (SigmaA-7049, purified) 8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin (ICN,Costa Mesa, Calif.) 100 mg/l; bialaphos (Meiji Seika K.K., Tokyo, Japan)1.5 mg/l for the first two weeks followed by 3 mg/l for the remainder ofthe time; pH 5.8] putting about 20 embryos onto each plate. The platesare sealed as described above and incubated in the dark at 28° C. forthe first two weeks of selection. The embryos are transferred to freshselection medium at two-week intervals. The tissue is subcultured bytransferring to fresh selection medium for a total of about 2 months.The herbicide-resistant calli are then “bulked up” by growing on thesame medium for another two weeks until the diameter of the calli isabout 1.5-2 cm.

For regeneration, the calli are then cultured on PHI-E medium [MS salts4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l, thiamine.HCl 0.1mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, Zeatin 0.5 mg/l,sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0 g/l, Indoleacetic acid (IAA,Sigma) 1.0 mg/l, Abscisic acid (ABA, Sigma) 0.1 mM, Bialaphos 3 mg/l,carbenicillin 100 mg/l adjusted to pH 5.6] in the dark at 28° C. for 1-3weeks to allow somatic embryos to mature. The calli are then cultured onPHI-F medium (MS salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl 0.1mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, nicotinic acid 0.5mg/l; sucrose 40.0 g/l; gelrite 1.5 g/l; pH 5.6] at 25° C. under adaylight schedule of 16 hrs. light (270 uE m-2sec-1) and 8 hrs. darkuntil shoots and roots develop. Each small plantlet is then transferredto a 25×150 mm tube containing PHI-F medium and grown under the sameconditions for approximately another week. The plants are transplantedto pots with soil mixture in a greenhouse. GUS+ events are determined atthe callus stage or regenerated plant stage.

For Hi-II a preferred optimized protocol was 0.5×109 cfu/mlAgrobacterium a 3-5 day resting step and no AgNO3 in the infectionmedium (PHI-A medium).

EXAMPLE 4

In situ Localization of End2 mRNA in Developing Maize Kernel

In situ hybridization was performed using the protocol of Jackson, D. P.(1991) In situ Hybridization in Plants, Molecular Plant Pathology: APractical Approach, D. J. Bowles, S. J. Gurr, and M. McPherson, eds.Oxford University Press, England, pp.63-74. Both a sense and antisenseprobe corresponding to a protein of the end2 cDNA were used. Probes werelabelled non-isotopically with digoxigenin and incubated with varioussections of 5 DAP and 9 DAP (days after pollination) kernels of maizeline CJ27 which had been fixed and embedded. Following extensive washingto remove unbound probe, sections were incubated with anti-digoxigeninalkaline phosphatase to detect areas of probe hybridization. For end2,mRNA was detected specifically with the antisense probe and restrictedto the aleurone layer of endosperm tissue. The sense control probe didnot hybridize.

EXAMPLE 5

Northern Analysis of Gene Expression in Vegetative Tissue and DevelopingKernels

Total RNA (10 μg) was size fractionated on a 1% formaldehyde agarose geland transformed to a nitrocellular membrane. Membranes were hybridizedunder stringent conditions with ³²P-labelled probes representing CDNAfragments of the various genes. After extensive washing to removeunbound probe, membranes were exposed on X-ray film. RNA samples wereobtained from vegetative tissues as well as developing maize kernels.

The end1 expression pattern showed no expression in vegetative tissues.End1 was predominantly expressed in early to mid-development wholekernel. Expression was also seen in isolated 7 DAP (days afterpollination) embryo, 10 DAP endosperm and 10 DAP pericarp.

The expression pattern of end2 likewise showed no expression invegetative tissue. End2 showed expression in 9-14 DAP whole kernel. End2also showed expression in isolated 7 DAP embryo, 10 DAP endosperm, and10 DAP pericarp.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

10 1 713 DNA Zea mays 1 ggctggtaaa aaccattatt aactttaaca tcgaatcaaaactgacaaat tttatacttt 60 cacagagcag cagaaattta tacaatatga ttgaatacaagatgtaggac ccgatggaga 120 gaattttttt gtctcctata tgcttgaata cccaacataatatcttcgca gcatactatc 180 tatctaatag aaaaattata atatagttaa atacttaagtagtatctagt ggatagaatt 240 caatatctca tacatgcatg aggagtaata tctactagacatgcaacata tttttatcta 300 tctaatagaa tatatataat aaagttaaat attatatgcatcacctacta tatataattt 360 gatatctttt agatgtataa gggactaaga ataatatctctagcacacat gcaatgcatt 420 atctatctaa atatattata taatagttaa atattaattatacgtagtct aaacctacat 480 ataagcctac ccatccccac ttagagatct cagtgtcacacatagaccat acatctcact 540 tcgccaagaa aatttcgtca acagttgaag ttatacccatggcaaaacta ctcttgggtt 600 tgctccttgc ccttgctatt ctagggacaa catcggctgctggttgtgta caagaagggc 660 gaattctgca gatatccatc acactggcgg ccgctcgagcatgcatctag agg 713 2 27 DNA Zea mays 2 tgttgggtat tcaagcatat aggagac 273 29 DNA Zea mays 3 ccatcgggtc ctacatcttg tattcaatc 29 4 1224 DNA Zeamays 4 tactataggg cacgcgtggt cgacggcccg ggctggtaaa aagtaattga acccaaaata60 tcatggtatg tttggtgaag acagtgatca gtgatttttt tatatctata tatatatcaa 120agatacttga ttttctagaa ggttcttttt gttgttttcc cttatgtttt tacgcatgat 180gcaattcttt ttgagaggtt tccgatgcat tgatgttatt gtattatctc ctatatatag 240gtcgacgtac attatgtatt gcaataacca gttaactgga tccagcttcg cttagttttt 300agtttttggc agaaaaaatg atcaatgttt cacaaaccaa atatttttat aacttttgat 360gaaagaagat caccacggtc atatctaggg gtggtaacaa attgcgatct aaatgtttct 420tcataaaaaa taaggcttct taataaattt tagttcaaaa taaatacgaa taaagtctga 480ttctaatctg attcgatcct taaattttat aatgcaaaat ttagagctca ttaccacctc 540tagtcatatg tctagtctga ggtatatcca aaaagccctt tctctaaatt ccacacccaa 600ctcagatgtt tgcaaataaa tactccgact ccaaaatgta ggtgaagtgc aactttctcc 660attttatatc aacatttgtt attttttgtt taacatttca cactcaaaac taattaataa 720aatacgtggt tgttgaacgt gcgcacatgt ctcccttaca ttatgttttt ttatttatgt 780attattgttg ttttcctccg aacaacttgt caacatatca tcattggtct ttaatattta 840tgaatatgga agcctagtta tttacacttg gctacacact agttgtagtt ttgccacttg 900tctaacatgc aactctagta gttttgccac ttgcctggca cgcgactcta gtattgacac 960ttgtatagca aataatgcca atacgacacc tggccttaca tgaaacatta tttttgacac 1020ttgtatacca tgcaacatta ccattgacat ttgtccatac acattatatc aaatatattg 1080agcgcatgtc acaaactcga tacaaagctg gatgaccctc cctcaccaca tctataaaaa 1140cccgagcgct actgtaaatc actcacaaca caacacatat cttttagtaa cctttcaata 1200ggcgtccccc aagaactagt aaac 1224 5 30 DNA Zea mays 5 acttatcaggctttggaggt cattctcaca 30 6 32 DNA Zea mays 6 tccatcagca tgagagagcctatggcaaac at 32 7 22 DNA Artificial Sequence primer 7 gtaatacgactcactatagg gc 22 8 19 DNA Artificial Sequence primer 8 actatagggcacgcgtggt 19 9 22 DNA Artificial Sequence commercial primer AP1 9gtaatacgac tcactatagg gc 22 10 19 DNA Artificial Sequence commericalprimer AP2 10 actatagggc acgcgtggt 19

What is claimed is:
 1. An isolated promoter that is capable of drivingtranscription in a seed-preferred manner, wherein said promotercomprises a nucleotide sequence set forth in SEQ ID NO:
 1. 2. Anexpression cassette comprising a promoter and a nucleotide sequenceoperably linked to said promoter, wherein said promoter is capable ofinitiating seed-preferred transcription of said nucleotide sequence in aplant cell, wherein said promoter comprises a nucleotide sequence setforth in SEQ ID NO:
 1. 3. A plant stably transformed with an expressioncassette comprising a maize promoter and a nucleotide sequence operablylinked to said promoter, wherein said promoter is capable of initiatingseed-preferred transcription of said nucleotide sequence in a plantcell, wherein said promoter comprises a nucleotide sequence set forth inSEQ ID NO:
 1. 4. An isolated promoter that is capable of drivingtranscription in a seed-preferred manner, wherein said promotercomprises a fragment of SEQ ID NO:1.
 5. An expression cassettecomprising a promoter and a nucleotide sequence operably linked to saidpromoter, wherein said promoter is capable of initiating seed-preferredtranscription of said nucleotide sequence in a plant cell, and whereinsaid promoter comprises a fragment of SEQ ID NO:1.
 6. A plant stablytransformed with an expression cassette comprising a maize promoter anda nucleotide sequence operably linked to said promoter, wherein saidpromoter is capable of initiating seed-preferred transcription of saidnucleotide sequence in a plant cell, and wherein said promoter comprisesa fragment of SEQ ID NO:1.